G-protein coupled signal transduction systems are responsible for receiving and processing information, thus enabling us to see, taste, smell and even to think. The core components of these systems are receptors, heterotrimeric GTP binding proteins (G proteins), and effector molecules. G proteins are composed of an alpha, beta, and gamma subunit. The alpha subunit has a guanine nucleotide binding site, and intrinsic GTPase activity. Signal transduction is initiated when an agonist interacts with its receptor forming a complex that is capable of facilitating the release of GDP from the G protein alpha subunit (G-alpha) so that GTP can bind and activate the transducer. The activated G protein subsequently regulates the activity of specific effector molecules until the GTP is hydrolyzed leading to deactivation of the G protein. G proteins can be irreversibly activated by non-hydrolyzable GTP analogs (ie. Gpp[CH2]p, Gpp[NH]p, and GTP-gamma-S). Although much is known about the individual components of these G protein-mediated signal transduction systems, there is much to be discovered about how these proteins interact during the signal transduction process in intact cells. The current hypothesis is that G protein activation by an agonist-receptor complex causes G-alpha to dissociate from the beta-gamma-heterodimer (G-beta-gamma), and that the individual components of this system move about independently of one another in the cell membrane. Consequently, signal transduction is thought to occur by a "random collision coupling mechanism".The fact that non-hydrolyzable GTP analogs reduce the affinity of G-alpha for G-beta-gamma has been used to buttress the hypothesis that GTP itself causes subunit dissociation when it activates G proteins in situ. Using surface plasmon resonance spectroscopy we have shown that in solution the affinity of G-alpha for G-beta-gamma varies with the guanine nucleotide that is bound to G-alpha. We have determined that the equilibrium binding constant for the subunits of the inhibitory G protein (Gi) is 10, 123, 235, or 433 nM when the nucleotide bound to Gi-alpha is GDP, Gpp[CH2]p, Gpp[NH]p or GTP-gamma-S respectively. It is clear that compared with GDP the non-hydrolyzable GTP analogs reduce the affinity of Gi subunits for each other, but the variability in their effects precludes claiming that they are representative of how GTP will effect Gi subunit affinity. The affinity of G protein subunits for each other is also affected by their environment. Although GTP-gamma-S can cause G-alpha to dissociate from G-beta-gamma in detergent containing solutions, we have shown that in cell membrane activation of the stimulatory G protein by GTP-gamma-S does not cause subunit dissociation. As with G protein subunits, there is increasing evidence that in cell membranes the other components involved in G protein mediated signal transduction form a more tightly associated complex than was previously hypothesized. We have begun bioluminescence resonance energy transfer experiments in order to determine if the components of this system are juxtaposed in the membrane. These studies will help us to understand how these critically important and pervasive signal transduction systems work so that we can improve the diagnosis and treatment of human diseases that occur when these systems malfunction.